Cumin Seed Oil Induces Oxidative Stress-Based Antifungal Activities on Fusarium graminearum

In this study, the antifungal activity of cumin seed oil (CSO) was tested on Fusarium graminearum. (i) Minimum inhibitory concentrations (MICs) and related concentrations (IC75, IC50, and IC25) were detected; (ii) toxicity was evaluated by a water-soluble tetrazolium salt-1 (WST-1) assay; (iii) genomic/epigenomic alterations were evaluated by the coupled restriction enzyme digestion-random amplification (CRED-RA) method; (iv) oxidative stress was investigated by CAT expression, catalase activity, and DCF-DA staining; (v) deoxynivalenol biosynthesis was evaluated by tri6 expression; (vi) and potential effects of CSO on wheat were tested by a water loss rate (WLR) assay. MIC, IC75, IC50 and IC25 values were detected at 0.5, 0.375, 0.25, and 0.125 mg mL−1. In WST-1 assays, significant decreases (p < 0.001) were detected. Genomic template stability (GTS) related to methylation differences ranged from 94.60% to 96.30%. Percentage polymorphism for HapII/MspI values were as 9.1%/15.8%. CAT (oxidative stress-related catalase) and tri6 (zinc finger motif transcription factor) gene expressions were recorded between 5.29 ± 0.74 and 0.46 ± 0.10 (p < 0.05). Increased catalase activity was detected (p < 0.05) by spectrophotometric assays. DCF-DA-stained (oxidative stressed) cells were increased in response to increased concentrations, and there were no significant changes in WLR values. It was concluded that CSO showed strong antifungal activity on F. graminearum via different physiological levels.


Introduction
Fusarium head blight (FHB) is one of the most destructive and devastating fungal diseases of small grain cereals worldwide.Epidemics result in reduction in crop quantity and quality.Consequently, economic losses reaching up to billions of dollars have been reported in many regions throughout the world [1][2][3].In addition to yield losses, mycotoxin contamination also occurs in wheat and barley fields where FHB present.Deoxynivalenol, nivalenol, and their acetylated derivatives have been reported as the predominating mycotoxins accumulated on small grain cereals infected with FHB causal agents, predominantly Fusarium graminearum [4][5][6][7].
Causal agents of FHB show variation due to several factors such as population dynamics of phytopathogens, climatic conditions, and crop rotations.However, F. graminearum sensu lato seems to be a predominating causal agent of FHB in particularly humid and semihumid regions [3,8,9].F. graminearum, with more than 10 members, has been accepted as a species complex nowadays and the global member of this species complex is F. graminearum sensu stricto [9][10][11].F. graminearum is a homothallic phytopathogen and it primarily infects wheat, maize, and barley worldwide.Also, this species complex produces several important mycotoxins such as deoxynivaleol, nivalenol, and zearalenone [4,12].F. graminearum is included in the list of most important phytopathogens worldwide [13].This fungus could also be considered as a model organism, and there are more than 100 genome assembly and annotation reports belonging to different strains deposited under GenBank, and at least three main genetic populations of F. graminearum in the northern hemisphere [14].
Development of disease resistant plant cultivars, usage of antagonistic microorganisms, and fungicide treatment have been widely used for in vitro and in planta studies to manage FHB.Each strategy has several disadvantages.Among these strategies, common fungicide treatment such as carbendazim and tetraconazole result in effective management against FHB and F. graminearum in many regions.However, it seems that fungicide resistance will become one of the most important topics in plant disease research in the near future [15][16][17][18][19][20][21][22][23][24].Especially, resistance to β-tubulin-and cyp51-targeting fungicides in Fusarium spp.seem to be even more important for agricultural sustainability for the near future.On the other hand, researchers have focused on working with alternative specific metabolites, which could be used in fighting against F. graminearum [25][26][27][28][29][30].These studies have been conducted using mainly phenotypic and transcript analysis.Further investigations could especially include epigenetics analysis, as well as fungicide and plant-derived essential oil mixtures for F. graminearum and related phytopathogens.
Since antimicrobial resistance remains an important problem for agriculture, researchers try to discover or develop novel antimicrobial compounds.Cumin (Cuminum cyminum L.) is well known for its beneficial effects, such as antibacterial, anti-cancerous and anti-diabetic effects, and natural essential oil extracts of CSO include useful and pharmaceutically important compounds, including cuminal and cuminic alcohol [31,32].However, detailed knowledge related to the mechanisms behind these useful effects of CSO is missing from the literature.In this study, the potential antifungal activity of cumin (Cuminum cyminum L.) seed oil (CSO) on F. graminearum was investigated.It was mainly aimed at revealing the (i) potential antifungal effects of CSO on F. graminearum and (ii) the mechanisms underlying the potential antifungal effects of CSO versus F. graminearum.In the current study, the effect of CSO on the reference strain PH-1 of F. graminearum was investigated in terms of phenotypic, genomic, epigenomic, transcript, and antioxidant levels for the first time.

Antimicrobial Assay and Cell Proliferation Test
A Fusarium graminearum PH-1 reference strain was used in this study.The fungal strain was grown on a potato dextrose agar (PDA) at 26 ± 2 • C for 7 days.CSO, including eugenol (0.5%) and limonene (0.4%) (W234300-Sigma, Saint Louis, MO, USA), was dissolved in ethanol.For minimum inhibition concentration (MIC) and related inhibition concentration determination, preliminary studies included different concentrations of CSO with logarithmic series of 2, 5, and 10 µg mL −1 .Mycelium discs (0.25 cm 2 ) from 7-day-old fresh fungal cultures were transferred onto a PDA medium with different concentrations of CSO (0, 0.125, 0.25, 0.375, and 0.5 mg mL −1 ) from CSO stocks dissolved in ethanol, with a concentration of 1mg mL −1 .The effect of the CSO was analyzed according to the linear growth rate measurements obtained on the 4th and 7th day of incubation.The growth inhibition as a percent (minimum inhibition concentration [MIC], growth inhibition at 75% [IC 75 ], half inhibition [IC 50 ], and growth inhibiton at 25% [IC 25 ]) was calculated in comparison to the control set.
The evaluation of cell proliferation was determined by a WST-1 (Roche, Basel, Switzerland) analysis.One cm 2 discs of control and experimental groups were dissolved in phosphate-buffered saline (PBS).A total of 10 µL WST-1 was added onto 100 µL cells suspended in PBS, and samples were incubated for 3 h at 28 • C in the dark.Spectrophotometric measurements were recorded at 450 and 620 nm wavelengths after incubation.The background radiation was excluded via the ∆ 450-620 nm calculation.The cytotoxicity level due to CSO treatment was evaluated by a comparison of absorbance values from the control and experiment sets.

Nucleic Acid Isolation and cDNA Synthesis
The genomic DNA (gDNA) from the control and experiment sets was extracted using a commercial genomic DNA isolation kit (Anatolia Geneworks, Istanbul, Turkey) following the experimental procedures provided by the manufacturer.The quality of gDNA was checked using 0.8% agarose gels.The concentration and purity of gDNA were analyzed by a spectrophotometer (Nanodrop-Thermo, Waltham, MA, USA).
The total RNA isolation from the control and experimental groups of F. graminearum PH-1 was performed using the monophasic reagent Hibrizol following the manufacturer's recommendations (Hibrigen, Istanbul, Turkey).The isolated RNA was analyzed with 0.8% agarose gels and spectrophotometric measurements.
The synthesis of cDNA from RNA molecules was carried out using the first strand cDNA synthesis kit (Takara, Shiga, Kansai, Japan).According to the recommendations from the manufacturer, 50 µM Oligo dT, 50 µM Random 6mer, 1× Buffer, 200 U Reverse Transcriptase enzyme, and total RNA molecules equal to 2 µg RNA were combined in 20 µL total volume.cDNA was synthesized by incubating samples at 37 • C for 20 min and 85 • C for 5 min.cDNAs were diluted by ¼ and then used in qPCR assays.

RAPD (Randomly Amplified Polymorphic DNA) and Coupled Restriction Enzyme
Digestion-Random Amplification (CRED-RA) Assays RAPD/CRED-RA techniques were used for the examination of the methylation status of the genome and template stability in response to the CSO treatment.The final concentrations of the components in the PCR assays were adjusted by 50 ng gDNA, 1× PCR buffer, 3 mM MgCl2, 0.4 mM dNTP mix, 10 pmol 10-mer primer, and 0.04 U µL −1 Taq DNA polymerase in a volume of 25 µL.In total, 23 common RAPD primers (Eurofins, Saint-Augustin, France) were used in this study (Table 1).Reaction was performed in two stages, following the pre-denaturation process at 94 • C for 2 min.Four loops in the first stage included incubation at 94 • C for 1.5 min, at 37 • C for 1.5 min, and at 72 • C for 3 min.The next 36 loops were performed for 1 min at 94 • C, 1 min at 37 ºC, and 2 min at 72 • C. The final elongation stage was carried out as 10 min at 72 • C. RAPD bands were run on 1.8% agarose gels and visualized under UV light [33].
The genomic template stability (GTS %) was calculated with the following formula: GTS = (1 − [a/n]) × 100, where a is the average number of polymorphic bands detected in each cumin-treated sample, and n is the number of total bands in the control set.Resolving power (RP) values for each primer were calculated using common formulas developed by Prevost and Wilkinson [34].
In CRED-RA assays, the gDNAs of the control and experimental groups were digested using HapII and MspI restriction endonucleases (Takara, Japan).The digestion reactions were combined in a total volume of 50 µL using 500 ng gDNA, 20 U HapII or MspI enzyme, 0.01% BSA, and 1× digestion buffer, and the mixture was incubated for 1 h 30 min at 37 • C. A stop reaction was carried out by adding 1× loading buffer and left at room temperature for 5 min.The digestion products were used for RAPD analysis as described before.The polymorphism value (%) was determined according to the formula: Polymorphism % = (5 − mC/5 + mC + C) × 100, where C is cytosine and mC is methylated cytosine [35].

Gene Expression Analysis
Fold changes in gene expression levels between control and experimental groups as a result of CSO treatment were determined by qPCR analysis.Genes associated with oxidative stress response (CAT: catalase, which has only one splice variant and the biological function was proved to be oxidative stress response [catalase_3: IPR018028/FGSG_06733]) and deoxynivalenol biosynthesis (tri6: a zinc finger transcription factor) were chosen as the target genes.β-tubulin was used as a housekeeping gene.qPCR assays were performed using the Sybr Green I fluorescent dye.qPCRs were conducted in a reaction volume of 20 µL containing 1× Sybr Green I mixture (Episozyme, Istanbul, Turkey), 5 pmol forward/reverse primers developed by Gazdagli et al. [25], and an amount of cDNA corresponding to 50 ng RNA.The cycling conditions, melting curve analysis, and fold change determination analysis were carried out as reported by Gazdagli et al. [25].

Intracellular ROS Detection by Fluorescence Microscopy
Changes related to oxidative stress that could occur in the experimental groups depending on the CSO treatment were revealed by fluorescence microscopy observations.For these purposes, carboxy methyl cellulose (CMC) liquid cultures were obtained.Up to seven 0.25 cm 2 fungal discs were transferred to a CMC broth medium of 25 mL and grown at 26 ± 2 • C for 7 days in a rotary shaker at 120 rpm. 1 × 10 5 spore mL −1 were used in fluorescence analysis.The presence of potential oxidative stress/reactive oxygen species (ROS) was investigated by 2 ′ ,7 ′ -dichlorofluorescin diacetate (DCF-DA) staining.Fungal cultures were incubated for 30 min at 25 • C using 4% formaldehyde and 0.1% Triton-X 100 to fix the samples.Fixed cells were washed twice with 1× PBS.Fluorescent staining was performed to determine the presence and status of ROS (DCF-DA; 5 µg mL −1 ) after washing.DCF-DA fluorescence staining was visualized with FITCH filter [28,29,36].ROS detection for potential oxidative stress response in F. graminearum was carried out via a PH2 condenser with excitation of 494 nm and emission of 518 nm.

Assessment of Catalase (CAT) Activity
Total protein from the control and experiment sets was extracted from 0.5 g of fresh mycelium using a common phosphate buffer protocol [37].Protein concentration was detected using bovine serum albumin standards series by using a bicinchoninic acid assay (BCA) protein assay kit following the manufacturer's recommendations (Atlas, Ankara, Turkey).An equalized protein concentration (1 mg) was used to take the kinetic measurement at 240 nm for detecting catalase activity.A reaction mix (112.2 µL phosphate buffer (50 mM NaH2PO4; pH 7.2) and 80 µL H 2 O 2 (40 mM)) were incubated at 30 • C for 2.5 min.After 6.8 µL protein extract was added to the reaction mix, the kinetic measurement was recorded at 10 s intervals for 2 min.The difference in absorbance through time (∆ 240 ) was used for inference of the catalase activity.Fold changes in CAT activity were calculated via normalization by control series.

Water Loss Rate Assay
The potential adverse effect of CSO on wheat was investigated via water loss rate (WLR) analysis.For this purpose, seeds of Triticum aestivum L. cv.Ceyhan 99 were used in WLR assays.The protocol provided by Suprunova et al. [38] was followed.A total of 10 seeds for each technical repeat were germinated on moist petri dishes at room temperature for seven days.The 7-day-old shoots with roots were transferred to plastic boxes containing soil.The plantlets were irrigated with distilled water and the CSO with 0.5 mg mL −1 for 7 days.The samples were incubated at 25 ± 2 • C. The expanded leaves of the 14-day-old plants were cut and the fresh weight (FW) was measured.The leaves were left on filter paper for 24 h, and then weight (W24) was measured.The leaves were left at 65 • C for 24 h and the dry weight (DW) was recorded.The WLR was calculated as following the formula: WLR (gh −1 g −1 DW) = (FW − W24)/(DW × 24).

Statistical Analysis
Each experimental procedure included two technical and three biological repeats.For statistical analyses, a t-test, one-way and two-way analysis of variance (ANOVA) with Tukey's post-test, normality tests (accompanied by Shapiro-Wilks test/Pearson's correlation matrix), and PCA analysis were carried out using GraphPad Prism 9.0 (Dotmatics, La Jolla, CA, USA) and R/R-Studio (Posit-PBC, Boston, MA, USA).The standard deviation for each experiment set was calculated via descriptive/column statistics.The confidence interval was determined at 0.05.

Gene Expression Analyses
Fold changes in CAT and tri6 expression were normalized in comparison to the βtubulin expression.Relative transcript abundance for the CAT gene in IC 25 , IC 50 , and IC 75 sets were recorded as 1.77 ± 0.25, 5.59 ± 0.74, and 6.13 ± 1.06, respectively (Figure 2A).There was no statistically significant difference between IC 25 treatment and the untreated control (p > 0.05), whereas the changes in IC 50 and IC 75 treatments were statistically significant (p < 0.001).Fold changes in the tri6 expression in IC 25 , IC 50 , and IC 75 treatments were calculated as 0.89 ± 0.07, 0.66 ± 0.06, and 0.46 ± 0.10, respectively (Figure 2B).Similar to CAT expression, there were no significant differences between IC 25 treatment and the control set in the tri6 expression (p > 0.05).The changes in IC 50 (p < 0.05) and IC 75 (p < 0.001) treatments were as statistically significant by one-way ANOVA analysis.

Fluorescence Microscopy Analysis
The presence of oxidative stress was evaluated via DCF-DA staining in the control, IC 25 , IC 50 , and IC 75 sets.ROS activity was not detected in most of the spores in the control set, whereas three experimental sets showed ROS activity under a fluorescent microscope.It was seen that the cells of IC 25 , IC 50 , and IC 75 sets were green colored (Figure 3).The number of spores (100 cell is equalized to "1" value) subjected to oxidative stress for the control, IC 25 , IC 50 , and IC 75 sets were recorded as 0.08 ± 0.01, 0.20 ± 0.03, 0.49 ± 0.04, and 0.59 ± 0.03, respectively.The changes in ROS activity were significantly different in IC 50 and IC 75 sets in comparison to the control set (p < 0.001), while no significant alteration was recorded in IC 25 via one-way ANOVA analysis.control (p > 0.05), whereas the changes in IC50 and IC75 treatments were statistically significant (p ˂ 0.001).Fold changes in the tri6 expression in IC25, IC50, and IC75 treatments were calculated as 0.89 ± 0.07, 0.66 ± 0.06, and 0.46 ± 0.10, respectively (Figure 2B).Similar to CAT expression, there were no significant differences between IC25 treatment and the control set in the tri6 expression (p > 0.05).The changes in IC50 (p ˂ 0.05) and IC75 (p ˂ 0.001) treatments were as statistically significant by one-way ANOVA analysis.

Fluorescence Microscopy Analysis
The presence of oxidative stress was evaluated via DCF-DA staining in the control, IC25, IC50, and IC75 sets.ROS activity was not detected in most of the spores in the control set, whereas three experimental sets showed ROS activity under a fluorescent microscope.It was seen that the cells of IC25, IC50, and IC75 sets were green colored (Figure 3).The number of spores (100 cell is equalized to "1" value) subjected to oxidative stress for the control, IC25, IC50, and IC75 sets were recorded as 0.08 ± 0.01, 0.20 ± 0.03, 0.49 ± 0.04, and 0.59 ± 0.03, respectively.The changes in ROS activity were significantly different in IC50 and IC75 sets in comparison to the control set (p ˂ 0.001), while no significant alteration was recorded in IC25 via one-way ANOVA analysis.

CAT Activity
The effect of CSO on the metabolization of the oxygen was examined by determining CAT activity alterations in the F. graminearum PH-1 strain.The CSO treatment led to significant differences between the control and the experiment sets via one-way ANOVA analysis.The values related to CAT activity (Δ240/1mg/min), 0.07 ± 0.01, 0.22 ± 0.01, 0.24 ± 0.03

CAT Activity
The effect of CSO on the metabolization of the oxygen was examined by determining CAT activity alterations in the F. graminearum PH-1 strain.The CSO treatment led to significant differences between the control and the experiment sets via one-way ANOVA analysis.The values related to CAT activity (∆ 240/1mg/min ), 0.07 ± 0.01, 0.22 ± 0.01, 0.24 ± 0.03 and 0.19 ± 0.03, were recorded in control, IC 25 (p < 0.01), IC 50 (p < 0.001), and IC 75 (p < 0.05) sets, respectively (Figure 4A).

CAT Activity
The effect of CSO on the metabolization of the oxygen was examined by determining CAT activity alterations in the F. graminearum PH-1 strain.The CSO treatment led to significant differences between the control and the experiment sets via one-way ANOVA analysis.The values related to CAT activity (Δ240/1mg/min), 0.07 ± 0.01, 0.22 ± 0.01, 0.24 ± 0.03 and 0.19 ± 0.03, were recorded in control, IC25 (p ˂ 0.01), IC50 (p ˂ 0.001), and IC75 (p ˂ 0.05) sets, respectively (Figure 4A).

WLR Analysis
No significant differences were detected between the WLR values of 14-day-old plantlets belonging to the control or the experiment sets (p > 0.05).Mean WLR values were recorded as 0.22 ± 0.01 and 0.20 ± 0.02 g h −1 g −1 for control and CSO treated sets (Figure 4B), respectively.No potential abiotic stress presence was detected due to CSO treatment in T. aestivum L. Ceyhan 99.

Statistical Analysis
Two-way ANOVA analyses were combined with Pearson's correlation matrix in order to reveal a correlation between different experimental procedures.By this way, data obtained from the WST-1 assay, DCF-DA staining analysis, catalase activity, CAT expression, and tri6 expression analysis were co-evaluated (Figure 4).In Pearson's correlation test, both negative and also positive correlations were found between different experimental procedures (Table 3).p Values were recorded between 0.004 and 0.258.The correlation efficiency values were detected between −0.986 and +0.955.It was clear that CAT activity and CAT expression presented a positive correlation.Similarly, positive correlation between cell viability and tri6 expression was recorded in the two-dimensional analysis (PC1: 74%, PC2: 11.4%, proportional variance).In contrast, negative correlation was also found in gene expression analysis.In PCAs, the control, IC 25 , IC 50 , and IC 75 sets were co-clustered in all different experimental procedures (Figure 5).recorded as 0.22 ± 0.01 and 0.20 ± 0.02 g h −1 g −1 for control and CSO treated sets (Figure 4B), respectively.No potential abiotic stress presence was detected due to CSO treatment in T. aestivum L. Ceyhan 99.

Statistical Analysis
Two-way ANOVA analyses were combined with Pearson's correlation matrix in order to reveal a correlation between different experimental procedures.By this way, data obtained from the WST-1 assay, DCF-DA staining analysis, catalase activity, CAT expression, and tri6 expression analysis were co-evaluated (Figure 4).In Pearson's correlation test, both negative and also positive correlations were found between different experimental procedures (Table 3).p Values were recorded between 0.004 and 0.258.The correlation efficiency values were detected between −0.986 and +0.955.It was clear that CAT activity and CAT expression presented a positive correlation.Similarly, positive correlation between cell viability and tri6 expression was recorded in the two-dimensional analysis (PC1: 74%, PC2: 11.4%, proportional variance).In contrast, negative correlation was also found in gene expression analysis.In PCAs, the control, IC25, IC50, and IC75 sets were co-clustered in all different experimental procedures (Figure 5).

Discussion
Plant-derived essential oils mixtures or specific compounds could show antimicrobial effects due to their valuable components.Particularly, these compounds could be an important part of an antagonistic effect on fungal phytopathogens [39,40].Up to now, many plant-derived essential oils, primarily as a methanol extracts, had been tested on F. graminearum or genetically closely related phytopathogenic fungal species in order to reveal their potential antifungal usage [28,39,[41][42][43].However, the previous studies mainly included revealing the composition of essential oil mixtures and determining the radial growth rate inhibition in fungi.The detailed and comprehensive data related to the mechanisms, which lie behind the potential antifungal activities of these plant-derived essential oils, are missing from the literature.Within this scope, the potential antifungal activity of cumin seed oil on physiological, genetics, epigenetics, and transcriptional levels of F. graminearum was evaluated.
Previous studies have shown that there are different ranges for inhibition values of radial growth rate in Fusarium spp.An approximately 50% growth inhibition due to aloe vera extracts treatment was recorded in F. oxysporum [42].Similarly, 1 µg µL −1 Haplophyllum tuberculatum essential oil extracts treatment resulted in a 49% radial growth inhibition in F. oxysporum.Singh et al. [39] reported that Foeniculum vulgare oil mixtures up to 6 µL led to 100% growth inhibition in F. graminearum.Perczak et al. [28] tested the potential antifungal activities of different plant-derived essential oil mixtures on F. graminearum and F. culmorum and they showed that there was great variation among minimum inhibition concentrations of different plant-derived essential oils.In this study, a relatively very low level of CSO was needed to repress the radial growth of F. graminearum.The MIC value for CSO (0.5 mg mL −1 ) was also relatively low in comparison to specific plant-derived essential oil compounds, such as α-thujone and camphor [25,26,29].Results obtained from this study yielded that the concentration to be used for in vitro tests of CSO is closely near to concentrations of specific fungicides [19,21,23,24].In addition to radial growth rate analysis, the potential growth inhibitory effects of CSO were also confirmed by using cell proliferation tests related to the WST-1 assay in this study.The presence of plant-derived essential oils led to toxicity in F. graminearum as reported by Teker et al. [29].
In recent years, several reports showed that plant-derived essential oil compounds or specific fungicides led to a high level of genomic and epigenomic alterations in phytopathogenic fungi [23,25,29].HapII and MspI enzymes were used in epigenomic analysis.HapII digestion is active in type I (CCGG; second C is methylated) and type II (CCGG; first C is methylated) methylation, whereas MspI enzyme is active for only type I and type III (CCGG; second C is methylated) methylation.Type IV (CCGG; first C is methylated) methylation includes no active digestion by HpaII and MspI [44,45].By using RAPD and CRED-RA methods, the potential genomic stability changes and type I-IV methylation presence were aimed to be revealed in this study.However, only a limited and low level of genomic instability was detected by the GTS and RP analyses.Similarly, type II and type III methylation changes of up to 15% polymorphisms were detected in the IC 25 , IC 50 , and IC 75 sets.A relatively stable methylation status was detected in experimental sets in comparison to previous studies, including the usage of specific plant-derived essential oil compounds [25,29].This low level of genomic and epigenomic changes due to CSO treatment (in comparison to specific plant-derived essential oil compounds or common fungicides) could arise from the complex or comprehensive content of the CSO, and some of the compounds present in the mixture could be effective in genomic and epigenomic stabilization.Further studies could include the effects of major and minor compounds of CSO mixtures on phytopathogenic fungi in terms of genomic or epigenomic alterations.
The changes in the expression of tri6 gene encoding the transcription factor of zinc finger motif related to trichothecene biosynthesis was also investigated in this study.Increased concentrations of CSO yielded a decreased expression of the tri6 gene.Down-regulation up to 54% in tri6 expression was recorded due to CSO treatment.This decrease in trichothecene biosynthesis-related gene expression was accordant with previous studies, including essential oil compound and fungicide treatment against Fusarium spp.[ [23][24][25]29].Moreover, negative correlations between tri6 expression and DCF staining and CAT expression were recorded.The findings in the current study provide preliminary data that CSO could be a potential repressor of trichothecene biosynthesis.However, in planta studies including CSO treatment could be useful for providing more detailed and comprehensive data in the near future.
The presence and adverse effects of oxidative stress in phytopathogenic fungi is one of the most important topics in plant pathology.Recent investigations showed that the presence and the level of oxidative stress in fungi (exposed to the stress factor[s]) affect important physiological processes such as mycotoxin biosynthesis and apoptosis destination in fungi [29,[46][47][48][49].In this study, potential oxidative stress presence in F. gramineraum in response to CSO treatment was investigated by qPCR, DCF-DA staining, and CAT activity assays.In comparison to previous studies, a moderate to high level of oxidative stress was detected via CSO treatment [2,29,[48][49][50].Each method independently showed that CSO treatment led to oxidative stress in the experiment sets.Moreover, CAT expression and the DCF-DA staining analysis were found to be positively correlated by posterior statistical analysis.The findings obtained from this study show that CSO strongly shows antifungal activities and represses radial growth on F. graminearum by oxidative stress in vitro for a limited part of the lifetime of fungi.Detailed antifungal activity of CSO or other plant-derived compounds on F. graminearum could be evaluated in terms of mycotoxin production and pigmentation processes.
This study, including data related to the hemi-biotrophic worldwide phytopathogen F. graminearum, provides preliminary and informative data for conducting similar studies in other fungal genera.The output of the study is of great importance in terms of revealing the potential of a new and natural antifungal compound that can be used in the fight against Fusarium spp.and related fungal phytopathogens' diseases.

Conclusions
Synthetic antifungal products, particularly common fungicides, have been used to manage and combat fungal diseases worldwide.However, they have detrimental effects on the environment such as the presence of fungicide resistant isolates and the accumulation of toxic waste.To avoid these undesirable effects, natural antifungal compounds are needed.This is the first report including physical, genetics, epigenetics, and transcriptional levels of investigations that show CSO could be considered as a potential antifungal agent against phytopathogenic fungi.Findings obtained from this study show that cumin seed oil overcomes, in particular, F. graminearum by oxidative stress formation in vitro.Further studies need in field investigations and mammalian culture tests to detect potential adverse effects of CSO on useful microorganisms and/or animals.

Figure 3 .
Figure 3. FITCH filter-captured profile of spores treated with different concentrations of cumin seed oil.(A): control set, (B): IC 25 set, (C): IC 50 set, and (D): IC 75 set at 20× magnification.Green-coloured cells show the presence of oxidative stress.

Figure 3 .
Figure 3. FITCH filter-captured profile of spores treated with different concentrations of cumin seed oil.(A): control set, (B): IC25 set, (C): IC50 set, and (D): IC75 set at 20× magnification.Green-coloured cells show the presence of oxidative stress.

Figure 5 .
Figure 5. Two-way ANOVA analysis (A), p values dendrogram for correlation status (B), and PCA profiling (C) of different experimental procedures carried out in this study.Control, IC25, IC50, and IC75 sets were the strains grown on PDA amended with 0, 0.125, 0.25, and 0.375 µg µL −1 cumin seed oil.

Figure 5 .
Figure 5. Two-way ANOVA analysis (A), p values dendrogram for correlation status (B), and PCA profiling (C) of different experimental procedures carried out in this study.Control, IC 25 , IC 50 , and IC 75 sets were the strains grown on PDA amended with 0, 0.125, 0.25, and 0.375 µg µL −1 cumin seed oil.

Table 1 .
RAPD primers and total amplicon numbers obtained from analysis.

Table 3 .
Pearson correlation matrix between experimental tests.Numeric data in columns are of Pearson r values.+ : positive correlation and − : negative correlation.